The AspB10 story

By the end of the 1980's Eli Lilly and Novo Nordisk both worked on short-acting insulin analogues. Initially, Novo Nordisk took the lead: their AspB10 analogue was the first short-acting insulin analogue tested in humans. However, while phase 2 trials in humans were ongoing, a 52-week study unexpectedly revealed a dose-dependent increase in mammary tumours in female rats. This put an abrupt halt to clinical testing in humans, but also led to extensive research to discover the reason for the mitogenic potential of this insulin analogue. Meanwhile, Eli Lilly profited from their competitor's setback: their insulin analogue insulin lispro became the first commercially available insulin analogue.

The development of AspB10

[1]Thinking about ways to increase the rapidity of insulin absorption, most researchers tried to alter the amino-acid composition in such a way that the insulin molecule would no longer self-associate to hexamers around a zinc atom. It was (correctly) hypothesized that such a monomeric insulin would have a more rapid absorption. The amino-acid at position B10, a histidine, was known to be involved in zinc binding and so it seemed rational to replace this with the negatively charged amino acid aspartic acid (hence the name AspB10).

The first tests of this new insulin analogue were promising. Absorption after subcutaneous was indeed twice as fast as absorption of human insulin, both in a pig model and in human pharmacokinetic studies. Clinically, a first study in humans demonstrated that postprandial glucose peaks decreased compared to regular human insulin, although (as was later found to be the case for all analogues) average glycaemia did not improve. Further studies however were cancelled when the potential for mitogenicity of the analogue became clear. Fortunately, the participants in the clinical studies had all been male, and at 10-year follow up, no serious side-effects had occurred.

Mitogenicity of AspB10

The investigation into the mitogenicity of AspB10 revealed several potential causative mechanisms:

AspB10 has a 200-400% increased binding affinity for the insulin receptor compared to human insulin in combination with a decreased rate of dissociation from the insulin receptor leading to a longer residence time on this receptor. Subsequent laboratory studies suggest that the longer the residence time of a certain analogue is, the higher its mitogenic potential. Biologically, insulin can act as a growth-promoting factor and some studies suggest that the longer residence time leads to a sustained intracellular phosphorylation signal which shifts the down-stream effects of the insulin receptor from metabolic to growth-promoting/mitogenic pathways.

AspB10 has an increased binding affinity for the Insulin-Like Growth Factor-1 (IGF-1) receptor compared to human insulin. Although this still constitutes a very low affinity in comparison with the binding affinity of IGF-1 itself, laboratory studies suggest that the higher the affinity of an insulin analogue for IGF-1, the higher the mitogenic potency of an insulin analogue. In contrast, insulin lispro, which was developed based on a structural similarity with IGF-1, and which has a higher IGF-1 receptor affinity as well, was not found to be associated with an increased carcinogenicity in either pre-clinical or clinical studies.

Which of these mechanisms is the main culprit, or indeed whether other yet undiscovered mechanisms play a part, is unclear. The data we have about why this side effect occurred are all obtained in cell or animal models, so it is hard to weigh the relevance of these findings in clinical practice. However, the AspB10 story has heightened the awareness that tampering with the insulin molecule is a delicate thing; nowadays all new insulin analogues have to be tested extensively for their mitogenic potential before studies in humans are allowed.